48 research outputs found
A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays
Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<30 ng/mL) determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings
Rapid Phenotypic Antibiotic Susceptibility Testing of Uropathogens Using Optical Signal Analysis on the Nanowell Slide
Achieving fast antimicrobial susceptibility results is a primary goal in the fight against antimicrobial resistance. Standard antibiotic susceptibility testing (AST) takes, however, at least a day from patient sample to susceptibility profile. Here, we developed and clinically validated a rapid phenotypic AST based on a miniaturized nanotiter plate, the nanowell slide, that holds 672 wells in a 500 nl format for bacterial cultivation. The multitude of nanowells allows multiplexing with a panel of six antibiotics relevant for urinary tract infections. Inclusion of seven concentrations per antibiotic plus technical replicates enabled us to determine a precise minimum inhibitory concentration for 70 clinical uropathogenic Escherichia coli isolates. By combining optical recordings of bacterial growth with an algorithm for optical signal analysis, we calculated Tlag, the point of transition from lag to exponential phase, in each nanoculture. Algorithm-assisted analysis determined antibiotic susceptibility as early as 3 h 40 min. In comparison to standard disk diffusion assays, the nanowell AST showed a total categorical agreement of 97.9% with 2.6% major errors and 0% very major errors for all isolate-antibiotic combination tested. Taking advantage of the optical compatibility of the nanowell slide, we performed microscopy to illustrate its potential in defining susceptibility profiles based on bacterial morphotyping. The excellent clinical performance of the nanowell AST, combined with a short detection time, morphotyping, and the very low consumption of reagents clearly show the advantage of this phenotypic AST as a diagnostic tool in a clinical setting
High-throughput screening for industrial enzyme production hosts by droplet microfluidics
A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its a-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in a-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains
Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays
New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level
High-Density Microwell Chip for Culture and Analysis of Stem Cells
With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field
Microfluidic device for generating a stepwise concentration gradient on a microwell slide for cell analysis
Understanding biomolecular gradients and their role in biological processes is essential for fully comprehending the underlying mechanisms of cells in living tissue. Conventional in vitro gradient-generating methods are unpredictable and difficult to characterize, owing to temporal and spatial fluctuations. The field of microfluidics enables complex user-defined gradients to be generated based on a detailed understanding of fluidic behavior at the lm-scale. By using microfluidic gradients created by flow, it is possible to develop rapid and dynamic stepwise concentration gradients. However, cells exposed to stepwise gradients can be perturbed by signals from neighboring cells exposed to another concentration. Hence, there is a need for a device that generates a stepwise gradient at discrete and isolated locations. Here, we present a microfluidic device for generating a stepwise concentration gradient, which utilizes a microwell slide's pre-defined compartmentalized structure to physically separate different reagent concentrations. The gradient was generated due to flow resistance in the microchannel configuration of the device, which was designed using hydraulic analogy and theoretically verified by computational fluidic dynamics simulations. The device had two reagent channels and two dilutant channels, leading to eight chambers, each containing 4 microwells. A dose-dependency assay was performed using bovine aortic endothelial cells treated with saponin. High reproducibility between experiments was confirmed by evaluating the number of living cells in a live-dead assay. Our device generates a fully mixed fluid profile using a simple microchannel configuration and could be used in various gradient studies, e.g., screening for cytostatics or antibiotics. (C) 2013 AIP Publishing LLC